Skeletal muscle development involves the withdrawal of myogenic stem cells from the cell cycle, the formation of multinucleated muscle fibers and the coordinate accumulation of the contractile proteins including alpha-actin, myosin heavy chain, myosin light chain, alpha-tropomyosin, troponins C,I and T, and M-creatine kinase. Although these genes are expressed in a tissue-specific fashion, the molecular regulatory mechanisms controlling their developmental expression remain largely unknown. As an initial step in determining how the contractile protein gene set is developmentally regulated, we have focused our efforts on identifying the transcriptional regulatory mechanisms controlling expression of the quail fast troponin I (TnI) gene (3). Previous studies demonstrated that the quail TnI gene, when introduced into the mouse multipotential cell line C3H10T1/2 (85, 39, 55), exhibits the correct developmental timing, tissue specificity and quantitative levels of gene expression associated with the endogenous mouse TnI gene (54, 53). Subsequent experiments have identified three regulatory regions that are responsible for this skeletal muscle-specific expression pattern. These include a 5' quantitative regulatory element, an intronic tissue-specific enhancer element and a 3' negative regulatory element. To fully characterize the TnI cis-acting regulatory regions and associated trans-acting factors, we propose to utilize in vitro mutagenesis strategies in conjunction with protein analysis and purification schemes. In addition, cDNA clones that express each of the TnI- specific trans-acting regulatory factors will be isolated by lambda gt11 library screening. These experiments will provide novel information on how TnI and other contractile protein genes are developmentally regulated. In addition, this study should produce new insights into the molecular mechanisms that are responsible for the differential transcription patterns observed in the developing embryo.